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Regulatory B Cells (B10 Cells) and
Regulatory T Cells Have Independent Roles
in Controlling Experimental Autoimmune
Encephalomyelitis Initiation and Late-Phase
Immunopathogenesis
Takashi Matsushita, Mayuka Horikawa, Yohei Iwata and
Thomas F. Tedder
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J Immunol published online 12 July 2010
http://www.jimmunol.org/content/early/2010/07/12/jimmun
ol.1001307
Published July 12, 2010, doi:10.4049/jimmunol.1001307
The Journal of Immunology
Regulatory B Cells (B10 Cells) and Regulatory T Cells
Have Independent Roles in Controlling Experimental
Autoimmune Encephalomyelitis Initiation and
Late-Phase Immunopathogenesis
Takashi Matsushita, Mayuka Horikawa, Yohei Iwata, and Thomas F. Tedder
M
ultiple sclerosis (MS) has been classically viewed as
a predominantly T cell-dependent autoimmune disease,
a feature shared by rheumatoid arthritis, systemic sclerosis, and type 1 diabetes. This conclusion derives from the finding
that the adoptive transfer of T cells from diseased animals can initiate disease symptoms in healthy recipients. By contrast, CD4+
regulatory T cells (Tregs) are critically important for limiting
T cell activation during MS and other autoimmune diseases (1, 2),
in part through their production of IL-10 (3). However, B cells also
regulate immune responses and can contribute to disease pathogenesis (4, 5) by functioning as cellular adjuvants for CD4+ T cell
activation (6) and through the production of cytokines that regulate
T cell function and inflammation (7). Furthermore, recent phase I
and II clinical trials in MS patients using depleting CD20 mAb
(rituximab) suggest that pan-mature B cell depletion has clinical
efficacy for the treatment of MS (8, 9), in addition to demonstrated
Department of Immunology, Duke University Medical Center, Durham, NC 27710
Received for publication April 21, 2010. Accepted for publication June 13, 2010.
This work was supported by Grants AI56363 and U54 AI057157 from the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense. T.M.
is supported by a fellowship from the Japan Society for the Promotion of Science.
The contents of this work are solely the responsibility of the authors and do not
necessarily represent the official views of the National Institutes of Health.
Address correspondence and reprint requests to Dr. Thomas F. Tedder, Box 3010, Department of Immunology, Room 353, Jones Building, Research Drive, Duke University
Medical Center, Durham, NC 27710. E-mail address: [email protected]
Abbreviations used in this paper: B10pro, B10 progenitor; BFA, brefeldin A; DC,
dendritic cell; EAE, experimental autoimmune encephalomyelitis; hCD19Tg, human
CD19 transgenic; L + PIM, LPS, PMA, and ionomycin, with monensin; MOG,
myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; Treg, T regulatory cell.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001307
efficacy for other autoimmune disorders (5). Despite these advances,
understanding of the complex mechanisms through which B cells
influence disease activity in humans and mice remains largely incomplete.
B cell-negative regulation of immune responses has been demonstrated in the mouse experimental autoimmune encephalomyelitis (EAE) model of human MS (10–12), and other mouse models
of autoimmunity and inflammation (7, 12–20). A relatively rare
spleen B cell subset with IL-10–dependent negative regulatory
functions has recently been identified that is predominantly contained within the phenotypically unique CD1dhighCD5+CD19high
spleen B cell subpopulation in mice (12, 17–19). A specific subset
of the CD1dhighCD5+ B cells can be induced to express cytoplasmic IL-10 following 5 h of in vitro stimulation with LPS, PMA,
and ionomycin, with monensin included in the cultures to block
IL-10 secretion (L + PIM stimulation). Given that multiple regulatory B cell subsets are likely to exist, as is now well recognized
for T cells, we have specifically labeled the IL-10–competent
CD1dhighCD5+ B cells as B10 cells because they appear to only
produce IL-10 and they are responsible for most B cell IL-10
production (21). B10 progenitor (B10pro) cells have also been
functionally identified in mice (5, 21). Spleen B10pro cells are
also found within the CD1dhighCD5+ B cell subpopulation, but
these cells require 48 h of in vitro stimulation with LPS or through
CD40 before they acquire the ability to express cytoplasmic IL-10
after 5-h stimulation with L + PIM (21). Although B10 cells
normally represent only 1–2% of spleen B cells, they dramatically
inhibit the induction of Ag-specific inflammatory reactions and
autoimmunity (12, 17).
Significant roles for B10 and B cells have been reciprocally
identified during the initiation and progression of EAE (12). Ma-
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Experimental autoimmune encephalomyelitis (EAE) is a T lymphocyte-mediated autoimmune disease of the CNS. Significant roles
for B cells and a rare IL-10–producing CD1dhighCD5+ regulatory B cell subset (B10 cells) have been identified during the initiation
and progression of EAE. Whether and how the regulatory functions of B10 cells and FoxP3+ T regulatory cells (Tregs) overlap or
influence EAE immunopathogenesis independently has remained unanswered. This study demonstrates that the number of
endogenous or adoptively transferred B10 cells directly influenced EAE pathogenesis through their production of IL-10. B10
cell numbers expanded quickly within the spleen, but not CNS following myelin oligodendrocyte glycoprotein35–55 immunization,
which paralleled B10 cell regulation of disease initiation. The adoptive transfer of myelin oligodendrocyte glycoprotein33–35sensitized B10 cells into wild-type mice reduced EAE initiation dramatically. However, B10 cells did not suppress ongoing
EAE disease. Rather, Treg numbers expanded significantly within the CNS during disease progression, which paralleled their
negative regulation of late-phase disease. Likewise, the preferential depletion of B10 cells in vivo during disease initiation
enhanced EAE pathogenesis, whereas Treg depletion enhanced late-phase disease. B10 cells did not regulate T cell proliferation
during in vitro assays, but significantly altered CD4+ T cell IFN-g and TNF-a production. Furthermore, B10 cells downregulated
the ability of dendritic cells to act as APCs and thereby indirectly modulated T cell proliferation. Thus, B10 cells predominantly
control disease initiation, whereas Tregs reciprocally inhibit late-phase disease, with overlapping B10 cell and Treg functions
shaping the normal course of EAE immunopathogenesis. The Journal of Immunology, 2010, 185: 000–000.
2
Materials and Methods
Cell preparation and immunofluorescence analysis
Single-cell leukocyte suspensions from spleens and peripheral lymph nodes
(paired axillary and inguinal) were generated by gentle dissection. Blood
mononuclear cells were isolated from heparinized blood after centrifugation over a discontinuous Lympholyte-Mammal (Cedarlane Laboratories,
Hornby, Ontario, Canada) gradient. CNS mononuclear cells were isolated
after cardiac perfusion with PBS, as described (23). Briefly, CNS tissues
were digested with collagenase D (2.5 mg/ml; Roche Diagnostics, Mannheim, Germany) and DNase I (1 mg/ml; Roche Diagnostics) at 37˚C for
45 min. Mononuclear cells were isolated by passing the tissue through
70-mm cell strainers (BD Biosciences, San Diego, CA), followed by Percoll gradient (70/37%) centrifugation. Lymphocytes were collected from
the 37:70% interface and washed.
Mouse CD20-specific mAb MB20-11 was used as described (24). FITC-,
PE-, PE Cy5-, PE Cy7-, or allophycocyanin-conjugated CD1d (1B1), CD3
(17A2), CD4 (H129.19), CD5 (53-7.3), CD8 (53-6.7), CD11b (M1/70),
CD11c (N418), CD19 (1D3), CD25 (PC61), B220 (RA3-6B2), and Thy1.1
(OX-7) mAbs were from BD Biosciences. PE-conjugated IL-10R mAb
(1B1.3a) was from BioLegend (San Diego, CA). Intracellular staining used
mAbs reactive with IL-10 (JES5-16E3), IFN-g (XMG1.2), TNF-a (MP6XT22), and FoxP3 (FJK-16s) (all from eBioscience, San Diego, CA) and
Cytofix/Cytoperm kits (BD Biosciences). For T cell intracellular cytokine
staining, lymphocytes were stimulated in vitro with PMA (50 ng/ml;
Sigma-Aldrich, St. Louis, MO) and ionomycin (1 mg/ml; SigmaAldrich), in the presence of brefeldin A (BFA, 1 ml/ml; eBioscience) for
5 h before staining. Background staining was assessed using nonreactive,
isotype-matched control mAbs (Caltag Laboratories, San Francisco, CA).
For two- to six-color immunofluorescence analysis, single-cell suspensions
(106 cells) were stained at 4˚C using predetermined optimal concentrations
of mAb for 20 min, as described (25). Blood erythrocytes were lysed after
staining using FACS lysing solution (BD Biosciences). Cells with
the forward and side light scatter properties of lymphocytes were analyzed
using a FACScan flow cytometer (BD Biosciences) or BD FACSCanto II
(BD Biosciences).
Mice and immunotherapy
C57BL/6 and IL-102/2 (B6.129P2-Il10tmlCgn/J) mice (26) were from
The Jackson Laboratory (Bar Harbor, ME). CD192/2 and human CD19
transgenic (hCD19Tg; h19-1 line) mice were backcrossed with C57BL/6
mice for 14 and 7 generations, respectively, as described (27, 28). TCRMOG
transgenic mice (Thy1.2+; provided by V. Kuchroo, Harvard Medical
School, Boston, MA) were crossed to C57BL/6.Thy1.1 mice to generate
Thy1.1-expressing T cells. All mice were bred in a specific pathogen-free
barrier facility and used at 6–12 wk of age.
To deplete B10 cells in vivo, sterile CD22 (MB22-10, IgG2c) or isotypematched mAbs (250 mg) were injected in 200 ml PBS through lateral tail
veins (29). To deplete CD4+CD25+FoxP3+ Tregs in vivo, denileukin diftitox (5 mg in 500 ml PBS; Ligand Pharmaceuticals, San Diego, CA) or PBS
was injected i.p. The Duke University Animal Care and Use Committee
approved all studies.
EAE induction
Active EAE was induced in 6- to 8-wk-old female mice by s.c. immunization with 100 mg myelin oligodendrocyte glycoprotein (MOG)35–55
peptide (MEVGWYRSPFSRVVHLYRNGK; NeoMPS, San Diego, CA)
emulsified in CFA containing 200 mg heat-killed Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) on day 0. Additionally, mice received
200 ng pertussis toxin (List Biological Laboratories, Campbell, CA) i.p. in
0.5 ml PBS on days 0 and 2. Clinical signs of EAE were assessed daily
with a 0- to 6-point scoring system, as follows: 0, normal; 1, flaccid tail;
2, impaired righting reflex and/or gait; 3, partial hind limb paralysis; 4,
total hind limb paralysis; 5, hind limb paralysis with partial fore limb
paralysis; 6, moribund state (11). Moribund mice were given disease severity scores of 6 and euthanized. Disease scores over the course of the
28-d experiments were totaled for each animal, and the mean for the
experimental group was expressed as a cumulative EAE score.
Histology
Following an initial perfusion with PBS, animals were perfused transcardially with 4% paraformaldehyde and spinal cords were removed.
Tissues were processed and blocked in paraffin wax. Two transverse sections of the thoracic and lumber spinal cord were stained with H&E and
Luxol Fast Blue. The number of inflammatory foci that contained at least
20 cells were counted in each H&E-stained section in a blinded fashion.
When foci coalesced, estimates were made of the number of foci. Areas of
demyelination were assessed for Luxol Fast Blue-stained sections. ImageJ
software (National Institutes of Health, Bethesda, MD) was used to manually trace the total cross-sectional area and the demyelinated area of each
section. Total demyelination was expressed as a percentage of the total
spinal cord area.
B10 cell analysis
Intracellular IL-10 expression was visualized by immunofluorescence staining and analyzed by flow cytometry, as described (17). Briefly, isolated
leukocytes or purified cells were resuspended (2 3 106 cells/ml) in complete medium (RPMI 1640 media containing 10% FCS, 200 mg/ml penicillin, 200 U/ml streptomycin, 4 mM L-glutamine, and 5 3 1025 M 2-ME
[all from Life Technologies, Carlsbad, CA]) with LPS (10 mg/ml, Escherichia coli serotype 0111:B4; Sigma-Aldrich), PMA (50 ng/ml; SigmaAldrich), ionomycin (500 ng/ml; Sigma-Aldrich), and monensin (2 mM;
eBioscience) for 5 h, in 48-well flat-bottom plates. In some experiments,
the cells were incubated for 48 h with an agonistic anti-mouse CD40 mAb
(1 mg/ml; HM40-3 mAb; BD Biosciences), as described (21). For IL-10
detection, FcRs were blocked with mouse FcR mAb (2.4G2; BD Biosciences) with dead cells detected using a LIVE/DEAD Fixable Violet Dead
Cell Stain Kit (Invitrogen-Molecular Probes, Carlsbad, CA) before cell
surface staining. Stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences), according to the manufacturer’s
instructions, and stained with PE-conjugated mouse anti–IL-10 mAb. Leukocytes from IL-102/2 mice served as negative controls to demonstrate
specificity and to establish background IL-10–staining levels.
Real-time RT-PCR analysis
Total RNA was extracted from cell sorter-purified B cells using Qiagen
RNeasy spin columns (Qiagen, Crawley, United Kingdom). Random hexamer primers (Promega, Madison, WI) and Superscript II RNase H reverse
transcriptase (Invitrogen, Carlsbad, CA) were used to generate cDNA. IL-10
transcripts were quantified by real-time PCR analysis using SYBR Green as
the detection agent. The PCR was performed with the iCycler iQ system
(Bio-Rad, Hercules, CA). All components of the PCR mix were purchased
from Bio-Rad and used according to the manufacturer’s instructions. Cycler
conditions were one amplification cycle of denaturation at 95˚C for 3 min,
followed by 40 cycles of 95˚C for 10 s, 59˚C for 1 min, and 95˚C for 1 min.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
ture B cell depletion in mice before EAE induction significantly
exacerbates disease symptoms, whereas B cell depletion during
EAE progression dramatically inhibits disease symptoms. B10
cell depletion from mice before disease initiation accounts for
exacerbated disease, which can be ameliorated by the adoptive
transfer of spleen CD1dhighCD5+ B cells. Similarly, IL-10 deficiency enhances the severity of EAE (22). Thereby, the balance
between opposing positive and negative regulatory B cell functions shapes the normal course of EAE immunopathogenesis.
Whether and how the regulatory functions of B10 cells and Tregs
overlap or influence EAE immunopathogenesis independently has
remained unanswered. To address this question, the regulatory
effects of adoptively transferring increasing numbers of naive
or EAE-sensitized B10 cells, or IL-10–deficient CD1dhighCD5+
B cells into wild-type mice at various stages of disease were
evaluated, in addition to depleting Tregs during both disease initiation and progression. Furthermore, we are the first to show in
this study that in vivo CD22 mAb treatment preferentially depletes
spleen B10 cells, which dramatically exacerbates EAE severity
during the initiation phase of disease. This study thereby demonstrates that B10 cells have different regulatory functions when
compared with Tregs, as they function at different time points
during EAE initiation and disease progression. Moreover, B10
cells directly influenced the production of proinflammatory cytokines by CD4+ T cells and suppressed the Ag-presenting function
of dendritic cells (DCs). Thereby, independent, but overlapping
B10 cell and Treg functions shape the normal course of EAE
immunopathogenesis.
B10 CELLS INHIBIT EAE
The Journal of Immunology
Specificity of the RT-PCR was controlled by the generation of melting
curves. IL-10 expression threshold values were normalized to GAPDH
expression using standard curves generated for each sample by a series
of four consecutive 10-fold dilutions of the cDNA template. For all reactions, each condition was performed in triplicate. Data analysis was performed using iQ Cycler analysis software. The sense IL-10 primer was 59GGTTGCCAAGCCTTATCGGA-39, and the antisense primer was 59-ACCTGCTCCACTGCCTTGCT-39. The sense GAPDH primer was 59-TTCACCACCATGGAGAAGGC-39, and the antisense primer was 59-GGCATGGACTGTGGTCATGA-39.
Lymphocyte subset isolation
Cell sorting and adoptive transfer experiments
Naive wild-type mice, wild-type mice with EAE, or IL-102/2 mice with
EAE were used as B cell donors. Splenic B cells were first enriched using
CD19 mAb-coated microbeads (Miltenyi Biotec). In addition, CD1dhigh
CD5+ and CD1dlowCD52 B cells were isolated using a FACSVantage SE
flow cytometer (BD Biosciences) with purities of 95–98%. After purification, 1 3 106 cells were immediately transferred i.v. into recipient mice.
In vitro T cell, B cell, and DC coculture assays
Splenic CD1dhighCD5+ or CD1dlowCD52 B cells from day 28 EAE mice
were purified by cell sorting. The sorted cell populations were stimulated
with agonistic CD40 mAb for 48 h, with LPS added during the final 5 h of
culture. TCRMOG CD4+ T cells were purified by MACS, and CFSE labeled. CFSE-labeled TCRMOG CD4+ T cells (1 3 106/ml) were cultured
alone or with CD40/LPS-stimulated CD1dhighCD5+ or CD1dlowCD52
B cells (1 3 106/ml) in the presence of MOG35–55 (25 mg/ml) for 72 h.
In additional experiments, splenic CD1dhighCD5+ or CD1dlowCD52
B cells from day 28 EAE mice were purified by cell sorting. The sorted cell
populations were stimulated with agonistic CD40 mAb for 48 h, with LPS
added during the final 5 h of culture. Splenic DCs from day 10 EAE mice
were purified by MACS and cultured (1 3 106/ml) with CD40 mAb/LPSstimulated CD1dhighCD5+ or CD1dlowCD52 B cells (1 3 106/ml) in the
presence of MOG35–55 (25 mg/ml) for 72 h. DCs were purified by cell
sorting after 72-h cocultures with B cells, and then cultured (5 3 104/ml)
with MACS-purified TCRMOG CD4+ T cells (2 3 105/ml) for 72 h.
Statistical analysis
All data are shown as means (6SEM). The significance of differences
between sample means was determined using the Student t test.
Results
B10 and Treg expansion during EAE
B10 cells and the spleen CD1dhighCD5+ B cell subpopulation are
significantly expanded in autoimmune prone mice (21). To determine whether B10 cells expand during EAE, B10 cell numbers
were quantified after L + PIM stimulation and staining for cytoplasmic IL-10 expression. After MOG35–55 immunization, spleen
CD1dhighCD5+ B cell frequencies and numbers were significantly
increased on days 7, 21, and 28 in contrast to naive mice (Fig. 1A).
B10 cell frequencies and numbers were also significantly increased
on days 7, 21, and 28 after MOG35–55 immunization (Fig. 1B).
Increased B cell IL-10 production paralleled B10 cell frequencies
(Fig. 1C), whereas immunizations with CFA alone had no effect on
B10 cell numbers (Fig. 1D). Thus, there was an initial increase in
B10 cell numbers and IL-10 transcripts following MOG35–55 immunization, which resolved, with a subsequent increase during EAE
disease onset and resolution.
Because Tregs negatively regulate EAE symptoms (2), spleen
CD25highFoxP3+CD4+ Treg numbers were also quantified. Treg
frequencies and numbers were only significantly higher by days
21 and 28 after MOG35–55 immunization in contrast to naive mice
(Fig. 1E). Thereby, B10 cell numbers increased during both the
initiation and late phases of EAE progression, whereas Treg
numbers were only increased during late-phase EAE.
B10 cell regulation of EAE
To determine whether quantitative differences in spleen B10 cell
numbers influenced EAE, disease initiation and progression were
compared in wild-type, CD19-deficient (CD192/2), and hCD19Tg
mice that overexpress CD19. Spleen CD1dhighCD5+ B cells were
present at similar frequencies and numbers in wild-type and
IL-102/2 mice (Fig. 2A), as described (17). In hCD19Tg mice,
CD1dhighCD5+ B cell frequencies and numbers were 4.4- and 1.6fold higher than in wild-type littermates, respectively. B10 cell
frequencies and numbers were also 5.6- and 1.8-fold higher in
hCD19Tg mice, respectively (Fig. 2B). By contrast, CD1dhigh
CD5+ B cell frequencies (93% decrease, p , 0.01) and numbers
(92% decrease, p , 0.01) were reduced in CD192/2 mice when
compared with wild-type mice, whereas B10 cell frequencies and
numbers were 62 and 76% lower, respectively (p , 0.01). Thus,
hCD19Tg and CD192/2 mice provided an optimal model system
for assessing the importance of B10 cell numbers during EAE.
EAE responses were assessed in MOG35–55-immunized CD192/2,
hCD19Tg, and wild-type mice. EAE symptoms first appeared on
about day 11 after immunization of wild-type mice and peaked on
about day 18, then declined gradually (Fig. 2C). By contrast, EAE
severity was significantly diminished in hCD19Tg mice (cumulative
EAE score, 17.5 6 4.7) when compared with wild-type mice
(38.1 6 5.0, p , 0.05). By contrast, EAE severity was significantly
enhanced in CD192/2 mice (62.5 6 3.3, p , 0.005), as reported
(31). Thus, enhanced or reduced B10 cell numbers in mice inversely
paralleled their disease symptoms.
To confirm that B10 cells regulated EAE responses in CD192/2
mice, spleen CD1dhighCD5+ or CD1dlowCD52 B cells were purified from naive wild-type mice and adoptively transferred (106
per recipient) into CD192/2 mice 24 h before immunization with
MOG35–55. CD1dhighCD5+ B cells were used for the adoptive
transfer experiments because this small subpopulation contains
both the B10 and B10pro cell subsets and can be identified without in vitro stimulation (17). Transferring CD1dhighCD5+ B cells
into CD192/2 mice significantly reduced EAE severity (cumulative EAE score, 40.6 6 5.0) to levels observed in wild-type mice
(37.2 6 5.6; Fig. 2D, left panel) when compared with CD192/2
mice (62.2 6 3.9, p , 0.01). By contrast, the adoptive transfer
of CD1dlowCD52 B cells into CD192/2 mice before EAE induction did not affect EAE severity (61.8 6 5.1; Fig. 2D, right panel).
Furthermore, the adoptive transfer of CD1dhighCD5+ B cells purified from IL-102/2 mice did not affect EAE severity (Fig. 2D).
Thus, B10 cells negatively regulated EAE development through
the production of IL-10, with increased B10 cell numbers significantly reducing disease severity.
Blocking CD22 ligand binding depletes B10 cells in vivo
To determine whether endogenous B10 cells regulate EAE,
methods were developed to preferentially deplete B10 cells. The
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MACS (Miltenyi Biotec, Auburn, CA) was used to purify lymphocyte
populations according to the manufacturer’s instructions. CD19 mAbcoated microbeads and CD4+ T cell isolation kits (Miltenyi Biotec) were
used to purify B cells and CD4+ T cells, respectively. When necessary, the
cells were enriched a second time using a fresh MACS column to obtain .95% cell purities, respectively. Spleen DCs were obtained as previously described, with minor modifications (30). Briefly, spleens were
minced and incubated with collagenase D (1 mg/ml) and DNase I (0.2
mg/ml) for 30 min at 37˚C. Cold EDTA was added to a final concentration
of 20 mM, and cell suspensions were incubated for 5 min at room temperature before filtering through nylon mesh to remove tissue and cell
aggregates. To enrich for cells with a low buoyant density, cellular suspensions were separated over a 30% BSA gradient and cells were collected
from the interface. After enrichment, DCs were isolated using CD11c
mAb-coated microbeads (Miltenyi Biotec).
3
4
B10 CELLS INHIBIT EAE
in vivo treatment of mice with mAbs that bind CD22 ligand binding
domains preferentially depletes splenic B cells with a marginal
zone phenotype, while leaving follicular B cells largely intact (29).
Because B10 and marginal zone B cells share some overlapping
cell surface markers (17), the ability of CD22 mAb (MB22-10) to
deplete B10 cells in vivo was quantified. Remarkably, CD22 mAb
treatment reduced both the frequency (86%) and number (90%) of
CD1dhighCD5+ spleen B cells by day 7 (Fig. 3A). CD22 mAb
treatment also reduced both the frequency (48%) and number
(54%) of spleen B10 cells by day 7, whereas control mAb treatment was without effect. Thereby, CD1dhighCD5+ B cells and
B10 cells could be preferentially removed without eliminating
the majority of spleen B cells.
B10 cells regulate EAE initiation, whereas Tregs regulate latephase EAE
The functional contributions of B10 cells to EAE initiation and
pathogenesis were measured after depleting B10 cells using CD22
mAb. First, mice were given CD22 mAb 7 d before and 0, 7, 14, and
21 d after EAE induction to deplete B10 cells before and during
EAE onset. B10 cell depletion did not accelerate disease onset, but
made disease severity significantly worse (cumulative EAE score,
52.3 6 4.4) in comparison with control mAb-treated littermates
(32.0 6 4.3; p , 0.01; Fig. 3B). In fact, 20% of the CD22 mAbtreated mice became moribund and were euthanized. Mice were
also given CD22 mAb on days 7, 14, and 21 after MOG35–55
immunization, which increased disease severity (44.8 6 2.08) in
comparison with control mAb-treated littermates (35.1 6 2.6).
B10 cell depletion in mice given CD22 mAb on days 14 and
21 after MOG35–55 immunization did not alter disease severity
(35.1 6 2.3) in comparison with control mAb-treated littermates
(30.9 6 3.3). B10 cell depletion in mice given CD22 mAb on
only day 21 did not alter disease severity. These findings argue
that B10 cell regulatory function is critical during disease initiation, but not after disease onset.
Treg depletion before MOG35–55 immunization can increase the
severity of EAE, whereas Tregs accumulate in the CNS during the
recovery phase of disease (32–35). Therefore, the functional
contributions of Tregs to EAE initiation and pathogenesis were
measured after their in vivo depletion. CD25 mAb was not used
for depleting Tregs because persistent mAb can deplete or inhibit
the expansion of activated T cells expressing CD25 and thereby
influence EAE pathogenesis independent of Treg depletion.
Rather, denileukin diftitox was used, a fusion protein of IL-2, and
diptheria toxin that binds to Tregs expressing high-affinity CD25
(36–38). Denileukin diftitox has limited effects on subsequently
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FIGURE 1. B10 cell and Treg numbers increase during EAE. A, CD1dhighCD5+ B cell frequencies and numbers increase during the course of EAE.
Splenocytes were isolated from mice before and 7, 14, 21, or 28 d after MOG35–55 immunization and analyzed for CD1d, CD5, and CD19 expression by
immunofluorescence staining with flow cytometry analysis. Representative results demonstrate the frequency of CD1dhighCD5+ B cells within the indicated
gates among total CD19+ B cells. Bar graphs indicate mean (6SEM) percentages and numbers of CD1dhighCD5+ B cells. B, IL-10+ B10 cell frequencies
and numbers increase during the course of EAE. Splenocytes were cultured with L + PIM for 5 h, stained with CD19 mAb, permeabilized, and stained using
IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10–producing cells within the indicated gates among total
CD19+ B cells. Bar graphs indicate mean (6SEM) percentages and numbers of B cells that produced IL-10. C, B cell IL-10 transcript expression during
EAE. RNA was isolated from splenic CD19+ B cells purified from mice before and 7, 14, 21, or 28 d after MOG35–55 immunization by MACS beads
(purities .99%). Values represent relative mean IL-10 transcript levels normalized to GAPDH transcript levels (6SEM) as quantified by real-time RT-PCR
analysis. D, CFA immunization does not affect IL-10+ B10 cell frequencies or numbers. Spleen B10 cell frequencies were examined before and after
immunization with CFA emulsified in an equal volume of PBS, as outlined in B. E, CD4+CD25+FoxP3+ T cell frequencies and numbers increase during the
course of EAE. Splenocytes were stained with CD4 and CD25 mAbs, permeabilized, and stained using FoxP3 mAb with flow cytometry analysis.
Representative results demonstrate the frequency of CD4+CD25+FoxP3+ T cells within the indicated gates among total CD4+ T cells. Bar graphs
indicate mean (6SEM) percentages and numbers of CD4+CD25+FoxP3+ T cells. A–E, Bar graphs indicate results from one of two independent
experiments with $5 mice in each group. Horizontal dashed lines are provided for reference to naive mice. Significant differences between means of
naive and immunized mice are indicated; pp , 0.05; ppp , 0.01.
The Journal of Immunology
5
activated effector T cells due to its short t1/2 in vivo. Spleen
CD25highFoxP3+CD4+ Tregs were maximally reduced 3 d after
denileukin diftitox injection, as previously reported (38), but
spleen CD25highFoxP3+CD4+ Tregs had returned by day 6 after
the initial injection. Therefore, multiple denileukin diftitox injections were given. Treg numbers were significantly reduced (60%)
in mice given denileukin diftitox 1, 4, and 7 d before Treg numbers were quantified (Fig. 3C). For Treg depletion before EAE
onset, mice were given denileukin diftitox 1, 4, 7, 10, and 13 d
after MOG35–55 immunization. This Treg depletion strategy
delayed EAE onset by 2 d, but the severity of disease symptoms
was not altered (cumulative EAE score, 29.6 6 5.5) in comparison
with PBS-treated littermates (33.6 6 5.7; Fig. 3D). For Treg depletion after EAE onset, mice were given denileukin diftitox
14, 17, 20, 23, and 26 d after EAE induction. Late-phase Treg
depletion worsened disease (54.0 6 4.4) significantly with 20% of
the treated mice becoming moribund in comparison with PBStreated littermates (35.6 6 5.0, p , 0.05). Thus, B10 cell function
was important for regulating EAE induction, whereas Treg function was important for regulating late-phase disease.
Tregs dominate the CNS after EAE development
Because B10 cells regulated EAE induction, whereas Treg function
regulated late-phase disease, the relative frequencies of B10 and
Tregs within the CNS, inguinal and axillary lymph nodes draining
the site of MOG immunization, and blood were compared. Within
the CNS, B10 cell frequencies relative to other B cells did not
change significantly during EAE development, although B10 cell
numbers were significantly increased on days 21 and 28 after
MOG35–55 immunization (Fig. 4A, left panels). By contrast,
CNS-infiltrating Treg frequencies relative to CD4+ T cells and
numbers were dramatically increased on days 7, 21, and 28 after
MOG35–55 immunization relative to naive mice (Fig. 4A, right
panels). Lymph node B10 cell frequencies were slightly increased
by 28 d after MOG35–55 immunization in contrast to naive mice,
whereas B10 cell numbers were significantly increased on days
21 and 28 after MOG35–55 immunization (Fig. 4B, left panels).
Treg frequencies within lymph nodes were significantly increased
on days 21 and 28 after MOG35–55 immunization in contrast to
naive mice, whereas Treg numbers were significantly increased
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FIGURE 2. B10 cell frequencies significantly influence EAE severity. A, CD1dhighCD5+ B cell frequencies and numbers in wild-type, hCD19Tg, CD192/2,
and IL-102/2 mice. Representative results demonstrate the frequency of spleen CD1dhighCD5+ B cells within the indicated gates among total CD19+ or
CD20+ B cells. Bar graphs indicate mean (6SEM) percentages and numbers of CD1dhighCD5+ B cells in one of two independent experiments with three
mice in each group. B, IL-10+ B10 cell frequencies and numbers in wild-type, hCD19Tg, CD192/2, and IL-102/2 mice. Representative results demonstrate
the frequency of spleen IL-10–producing cells within the indicated gates among total CD19+ or CD20+ B cells. Bar graphs indicate mean (6SEM)
percentages and numbers of B cells that produced IL-10. A and B, Values in each group represent results from six mice from two pooled independent
experiments. Significant differences between means from wild-type and other mouse groups are indicated; pp , 0.05; ppp , 0.01. C, EAE progression and
severity in wild-type, CD192/2, and hCD19Tg mice immunized with MOG35–55 on day 0 and scored daily thereafter for EAE disease severity. Values
represent mean (6SEM) EAE clinical scores from $5 mice in each group, with similar results obtained in two independent experiments. Significant
differences between wild-type and mutant mice groups are indicated; pp , 0.05 (wild-type versus hCD19Tg mice); †p , 0.05 (wild-type versus CD192/2
mice). D, Adoptively transferred CD1dhighCD5+ B cells reduce EAE severity in CD192/2 mice. Splenic CD1dhighCD5+ or CD1dlowCD52 B cells were
purified from naive wild-type or IL-102/2 mice by cell sorting. Wild-type and CD192/2 recipient mice were given either PBS, purified CD1dhighCD5+, or
CD1dlowCD52 B cells 1 d (arrowheads) before MOG35–55 immunizations. Values represent mean (6SEM) results from $5 mice in each group, with similar
results obtained in two independent experiments. Significant differences between the means of EAE clinical scores are indicated; pp , 0.05 (♦ versus )).
6
B10 CELLS INHIBIT EAE
during all stages of EAE (Fig. 4B, right panels). In blood, B10 cell
frequencies and numbers were significantly increased on 28 d after
MOG35–55 immunization (Fig. 4C, left panels). However, circulating Treg frequencies and numbers were not changed during the
course of EAE (Fig. 4C, right panel). As a consequence, Tregs far
outnumbered B10 cells within CNS tissues after EAE development, although the relative numbers of B10 and Tregs within the
spleen and lymph nodes were essentially unchanged during the
course of EAE (Fig. 4D). By contrast, blood B10 cell numbers
increased gradually relative to Tregs during EAE progression.
Thus, B10 cells were far more prevalent than Tregs within the
CNS before disease initiation, with Tregs dominating during latephase EAE.
Adoptively transferred B10 cells inhibit EAE progression
FIGURE 3. B10 cells and Tregs cooperatively regulate EAE severity. A,
B10 cell depletion in vivo. Spleen CD1dhighCD5+ and IL-10+ B cell
frequencies were determined in wild-type mice 7 d after MB22-10 or
control mAb treatments (250 mg/mouse). Upper panels, Representative
results demonstrate the frequency of CD1dhighCD5+ B cells within the
indicated gates among total CD19+ B cells. Lower panels, Representative
results demonstrate the frequencies of IL-10+ cells within the indicated
gates among total CD19+ B cells. Bar graphs indicate mean (6SEM)
percentages and numbers of CD1dhighCD5+ or IL-10+ B cells from six
mice in each group from two pooled independent experiments. Significant differences between MB22-10 or control mAb treatment groups are
indicated; ppp , 0.01. B, Early B10 cell deletion exacerbates EAE disease
severity. Mice were treated with MB22-10 (d) or control (s) mAb
(250 mg, arrowheads) from day 27 (days 27, 0, 7, 14, 21), day 7 (days
7, 14, 21), day 14 (days 14, 21), or day 21, as indicated. Values represent
mean (6SEM) EAE clinical scores from $5 mice in each group, with
similar results obtained in two independent experiments. Significant differences between MB22-10 and control mAb-treated groups are indicated;
pp , 0.05. C, Treg deletion. Representative results demonstrate spleen
CD4+CD25+FoxP3+ Treg frequencies within the indicated gates among
total CD4+ T cells and Treg numbers in wild-type mice 7 d after initial
denileukin diftitox or PBS treatments. Denileukin diftitox was administrated 1, 3, or 6 d before analysis or 1, 4, and 7 d before analysis, as
indicated. Bar graphs indicate mean (6SEM) percentages and numbers
of CD4+CD25+FoxP3+ Tregs from six mice in each group from two pooled
independent experiments. Significant differences between denileukin diftitox or PBS treatment groups are indicated; pp , 0.05; ppp , 0.01. D,
Late Treg deletion exacerbates EAE disease severity. Mice were treated
with denileukin diftitox (d) or PBS (s) before (days 1, 4, 7, 10, 13,
arrowheads) or after EAE onset (days 14, 17, 20, 23, 26, arrowheads).
Values represent mean (6SEM) EAE clinical scores from $5 mice in
each group, with similar results obtained in two independent experiments.
Significant differences between denileukin diftitox and PBS treatment
groups are indicated; pp , 0.05.
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The effect of increasing B10 cell numbers in wild-type mice on
EAE responses was assessed using adoptive transfer experiments.
Spleen CD1dhighCD5+ B cells and CD1dlowCD52 B cells were
purified from naive mice (Fig. 5A, left panels), and transferred
into mice that were immunized with MOG35–55 24 h after the
transfer. In these adoptive transfer experiments, a total of 1.23 3
105 of the transferred CD1dhighCD5+ B cells or 0.05 3 105 of
the transferred CD1dlowCD52 B cells expressed cytoplasmic
IL-10 after 5 h in vitro stimulation with L + PIM in two independent experiments. In one of two experiments with identical results,
the adoptive transfer of CD1dhighCD5+ B cells from naive mice
reduced disease severity (cumulative EAE score, 30.8 6 2.5) in
comparison with mice given CD1dlowCD52 B cells (42.6 6 4.5),
but this effect was not statistically different from EAE induction
or progression in PBS-treated littermates (36.6 6 3.8; Fig. 5C,
upper-left panel). Therefore, spleen CD1dhighCD5+ B cells and
CD1dlowCD52 B cells were purified from mice with EAE on
day 28 after MOG35–55 immunization. The frequency of B10 cells
within the CD1dhighCD5+ B cell subsets was comparable when the
donors were either naive mice (Fig. 5A) or mice with EAE (day
28; Fig. 5B, middle panels). Thereby, a total of 1.27 3 105 of
the transferred CD1dhighCD5+ B cells or 0.05 3 105 of the
transferred CD1dlowCD52 B cells expressed cytoplasmic IL-10
after 5-h stimulation with L + PIM in two independent experiments. In one of two experiments with identical results, transferring CD1dhighCD5+ B cells purified from mice with EAE
significantly reduced EAE severity (31.6 6 4.1; days 13–18,
p , 0.05) in recipients when compared with littermates given
CD1dlowCD52 B cells (51.4 6 4.5) or PBS-treated littermates
The Journal of Immunology
7
(43.4 6 4.7; Fig. 5C, upper-second panel). Thus, providing naive
mice with B10 cells from Ag-primed mice significantly reduced
EAE symptoms, whereas more modest effects were obtained when
the B10 cells were isolated from naive mice.
The spleen CD1dhighCD5+ B cell subset contains B10pro cells
that become competent to express IL-10 after in vitro CD40 engagement for 48 h to induce their maturation into IL-10–competent
B10 cells (21). The CD1dhighCD5+ B cell subset from naive mice
or mice with EAE (day 28) normally contains 12% IL-10+ B10
cells after 5 h of L + PIM stimulation (Fig. 5A, 5B). However, after
48 h of agonistic CD40 mAb stimulation, 40% of the purified
CD1dhighCD5+ B cells expressed cytoplasmic IL-10, whereas ,2%
of purified CD1dlowCD52 B cells produced IL-10 (Fig. 5B, right
panels). Thereby, the frequencies of IL-10–competent B10 cells
among CD1dhighCD5+ B cells can be enhanced significantly by
B10pro cell maturation in vitro.
For subsequent adoptive transfer experiments, CD1dhighCD5+
and CD1dlowCD52 B cells were purified from mice with EAE and
stimulated with agonistic CD40 mAb for 48 h, with LPS added
during the final 5 h of culture. In these adoptive transfer experiments, a total of 3.95 3 105 of the transferred CD1dhighCD5+
B cells or 0.19 3 105 of the transferred CD1dlowCD52 B cells
expressed cytoplasmic IL-10 in vitro after 5-h stimulation with
L + PIM in two independent experiments. In one of two experi-
ments with identical results, transferring CD40/LPS-stimulated
CD1dhighCD5+ B cells dramatically inhibited EAE progression
(cumulative EAE score, 15.6 6 6.9; days 12–22, p , 0.05) in
comparison with PBS-treated littermates (42 6 4, p , 0.05),
whereas CD1dlowCD52 B cells did not (42 6 6; Fig. 5C, lowerleft panel). However, inhibition of disease was only observed
when the transferred cells were given before MOG35–55 immunization of recipients, but not on days 7 or 14 after immunization
(Fig. 5C, lower-second and lower-third panels). Furthermore,
CD40/LPS-stimulated CD1dhighCD5+ B cells purified from IL102/2 mice with EAE (day 28) did not affect EAE severity in
wild-type recipients (Fig. 5C, lower-right panel). Thus, CD40/
LPS-stimulated CD1dhighCD5+ B cells optimally inhibited EAE
initiation in an IL-10–dependent manner.
Adoptively transferred B10 cells inhibit leukocyte infiltration
into the CNS
B cell depletion before MOG35–55 immunization exacerbates EAE
and results in higher numbers of CD4+ T cells within the CNS,
whereas B cell depletion following disease initiation reduces
CD4+ T cell infiltration into the CNS (12). Whether the adoptive
transfer of B10 cells from EAE mice inhibited T cell infiltration into the CNS of MOG35–55-immunized mice was therefore
assessed. CNS tissues were collected on day 18 from groups of
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FIGURE 4. Changes in B10 and Treg frequencies and numbers within tissues during the
course of EAE. Mononuclear cells were isolated from CNS tissue (A), inguinal and axillary lymph nodes (B) draining the site of MOG
immunization, or blood (C) before and 7, 14,
21, or 28 d after MOG35–55 immunizations (left
panels). Representative results demonstrate the
frequencies of IL-10–producing cells after L +
PIM stimulation for 5 h within the indicated
gates among total CD19+ B cells, as well as
CD4+CD25+FoxP3+ Treg frequencies among
total CD4+ T cells. Bar graphs indicate mean
(6SEM) frequencies and numbers of B cells
that produced IL-10 or CD25+FoxP3+CD4+
Tregs from six mice in each group from two
pooled independent experiments. Horizontal
dashed lines are provided for reference to
naive mice. D, Tregs dominate CNS tissues
after EAE development. Bar graphs indicate
mean (6SEM) percentages of IL-10+ B10
cells relative to total IL-10+ B10 cells plus
Tregs within various tissues. A–D, Significant
differences between means of naive mice and
mice with EAE are indicated; pp , 0.05;
ppp , 0.01. Similar results were obtained in
at least two independent experiments.
8
B10 CELLS INHIBIT EAE
mice that had been given PBS, or CD40/LPS-stimulated CD1dhigh
CD5+ or CD1dlowCD52 B cells from EAE mice (day 28), and
were examined by immunofluorescence staining of lymphocytes
or quantitative microscopy. Remarkably, CD40/LPS-stimulated
CD1dhighCD5+ B cell transfers significantly reduced both Treg
and CD4+ T cell numbers within the CNS, whereas CD1dlow
CD52 B cells were without effect (Fig. 6A). Spleen Treg and
CD4+ T cell numbers were not changed among these groups. As
quantified by microscopy, CD40/LPS-stimulated CD1dhighCD5+
B cell transfers reduced leukocyte infiltration (84% decrease thoracic, 84% lumbar; Fig. 6B) and significantly reduced demyelination (93% decrease thoracic, 88% lumbar; Fig. 6C) within CNS
tissues when compared with mice given PBS (p , 0.01; Fig. 6D).
Thus, adoptively transferred CD1dhighCD5+ B cells had profound
effects on Treg and leukocyte infiltration into the CNS.
B10 cells do not inhibit T cell proliferation, but regulate their
cytokine production
In vitro T cell–B cell coculture systems were developed to determine how B10 cells could regulate T cell-mediated autoimmune disease in vivo. First, purified spleen CD1dhighCD5+
B cells or CD1dlowCD52 B cells were stimulated with agonistic
CD40 mAb (48 h) and LPS (last 5 h of culture), washed extensively, and added to cultures containing MOG35–55 and CFSElabeled CD4+ T cells from TCRMOG transgenic mice whose
CD4+ T cells respond to MOG35–55 peptide (39). CFSE dilution
was assessed 72 h later as a marker for T cell proliferation.
TCRMOG CD4+ T cells cultured without B cells or without
MOG35–55 added to the cultures did not proliferate (Fig. 7A, data
not shown). However, TCRMOG CD4+ T cells cultured with either
CD1dhighCD5+ or CD1dlowCD52 B cells proliferated equally well
in response to MOG35–55. Thus, B10 cells did not regulate T cell
proliferation in these in vitro assays. However, when the TCRMOG
CD4+ T cells were cocultured with B cells in the presence of
MOG35–55, there were significant differences in T cell cytokine
induction observed following PMA, ionomycin, and BFA stimulation during the final 5 h of culture. TCRMOG CD4+ T cells
cultured with CD1dhighCD5+ B cells had dramatically reduced
IFN-g and TNF-a production when compared with TCRMOG
CD4+ T cells that were cultured with CD1dlowCD52 B cells
(Fig. 7B). These changes depended on B cell IL-10 production,
because CD1dhighCD5+ B cells from IL-102/2 mice did not affect
IFN-g or TNF-a production by TCRMOG CD4+ T cells. IL-10
production by TCRMOG CD4+ T cells was not significantly
changed in these culture systems. Thus, B10 cell IL-10 can regulate Ag-specific T cell cytokine production.
B10 cells regulate Ag presentation by DCs in vitro
IL-10R expression is heterogeneous among cells of the immune
system (40). Therefore, IL-10R expression by splenic DCs, macrophages, B cells, CD4+ T cells, and CD8+ T cells from wild-type
mice was assessed. IL-10R expression was highest on DCs and
macrophages, with modest expression by CD4+ T cells, CD8+
T cells, and B cells (Fig. 7C). IL-10R expression was not increased on any of these leukocyte subpopulations during the
course of EAE (day 7).
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FIGURE 5. The adoptive transfer of MOG35–55sensitized B10 cells can reduce EAE disease severity in wild-type mice. A and B, Representative
B10 cell purification results for adoptive transfer
experiments. Purified spleen B cells from naive
mice or mice with EAE (day 28) were separated
into CD1dhighCD5+CD19+ and CD1dlowCD52
CD19+ B cell populations. The isolated cells
were cultured with L + PIM for 5 h, or were
cultured with agonistic CD40 mAb for 48 h with
L + PIM added during the final 5 h of culture.
B10 cell frequencies in the stimulated cell cultures were determined by immunofluorescence
staining with flow cytometry analysis. C, The
adoptive transfer of purified spleen CD1dhigh
CD5+ B cells reduces EAE disease severity.
Wild-type recipient mice were given either PBS,
CD1dhighCD5+ B cells, or CD1dlowCD52 B cells
from naive wild-type mice, mice with EAE
(day 28), or IL-102/2 mice with EAE (day 28)
1 d before MOG35–55 immunizations or 7 or
14 d after MOG35–55 immunization, as indicated
(arrowheads). In some cases, the purified B cell
populations were stimulated as indicated with
agonistic CD40 mAb for 48 h, with LPS added
during the final 5 h of culture to induce B10pro
cell maturation and thereby expand B10 cell
frequencies. Values represent means (6SEM)
from $5 mice in each group, with similar results
obtained in two independent experiments. Significant differences between the means of EAE clinical scores are indicated; pp , 0.05 (♦ versus s).
The Journal of Immunology
9
Because high IL-10R expression by DCs may render them more
sensitive to the regulatory effects of IL-10 than CD4+ T cells, a role
for B10 cells in regulating DC activation of CD4+ T cells was
assessed. Briefly, purified splenic CD1dhighCD5+ or CD1dlow
CD52 B cells from mice with EAE (day 28) were stimulated with
agonistic CD40 mAb for 48 h, with LPS added during the final 5 h
of culture. Purified splenic DCs from mice with EAE (day 10)
were then cocultured with the CD40/LPS-stimulated CD1dhigh
CD5+ or CD1dlowCD52 B cells in the presence of MOG35–55.
After 72 h of culture, the DCs were purified by cell sorting and
cultured with TCRMOG CD4+ T cells for 72 h. The TCRMOG CD4+
T cells were then stained for CD4 and Thy1.1 expression and
analyzed for CFSE dilution. In cultures in which the TCRMOG
CD4+ T cells were cultured with DCs cocultured with CD1dhigh
CD5+ B cells, the intensity of CFSE staining was significantly
higher and the percentage of dividing TCRMOG CD4+ T cells
was significantly reduced when compared with DCs cultured
with CD1dlowCD52 B cells (p , 0.05; Fig. 7D). By contrast,
culturing DCs with CD1dhighCD5+ B cells from IL-102/2 mice
did not affect their Ag-presenting ability in this T cell activation
assay. Thus, IL-10–competent CD1dhighCD5+ B10 cells were able
to regulate the Ag-presenting capability of DCs.
Discussion
This study reveals that B10 cells predominantly reduce disease
severity during EAE initiation through the production of IL-10,
whereas Tregs reciprocally inhibit late-stage EAE immunopathogenesis (Fig. 3). Remarkably, the early expansion in B10 cell
numbers and IL-10 production following MOG35–55 immunization
parallels B10 cell regulation of disease initiation (Fig. 1A–C),
whereas Treg expansion during disease progression parallels their
negative regulation of late-stage disease (Fig. 1E). The current
study also demonstrates that numbers of endogenous or adoptively
transferred B10 cells directly influence the outcome of EAE pathogenesis. Specifically, mice with decreased B10 cell numbers
exhibited enhanced disease severity relative to wild-type mice,
whereas mice with enhanced B10 cell numbers had reduced
EAE severity (Fig. 2). Likewise, the preferential depletion of
B10 cells in vivo by CD22 mAb treatment enhanced EAE
pathogenesis (Fig. 3A, 3B). By contrast, the adoptive transfer of
Ag-sensitized B10 cells into naive recipients before MOG35–55
immunizations inhibited EAE pathogenesis through the production of IL-10, whereas increasing B10 cell numbers in mice
exhibiting disease symptoms were without significant effect
(Fig. 5). Thus, regulatory B10 cells and Tregs have independent
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FIGURE 6. Treg and leukocyte infiltration into
the CNS following the adoptive transfer of B10
cells. Wild-type recipient mice were given PBS,
or either CD40/LPS-stimulated CD1dhighCD5+
or CD1dlowCD52 B cells from mice with EAE
(day 28) 1 d before MOG35–55 immunizations,
as in Fig. 5C. A, Mononuclear cells within CNS
tissues or spleen 18 d after MOG35–55 immunizations. Bar graphs indicate mean (6SEM) numbers
of CD4+ T cells or frequencies/numbers of CD4+
CD25+FoxP3+ Tregs among total CD4+ T cells
(n $ 5 mice per group). Significant differences
between groups of mice receiving CD1dhighCD5+
B cells or PBS are indicated; ppp , 0.01. Similar
results were obtained in at least two independent
experiments. B–D, EAE histopathology following
the adoptive transfer of B10 cells. Representative
lumbar spinal cord sections were harvested 18 d
after MOG35–55 immunizations (n $ 5 mice per
group) with B, inflammation (H&E staining) and
C, demyelination (Luxol Fast Blue staining) demonstrated. Scale bar is 0.5 mm. B, Arrowheads
indicate inflammatory foci. C, Yellow traced areas
indicate demyelination. D, Bar graphs indicate
mean (6SEM) numbers of inflammatory foci
and percentages of spinal cord sections that were
demyelinated, with significant differences between
groups of mice receiving CD1dhighCD5+ B cells or
PBS indicated; ppp , 0.01. Similar results were
obtained in at least two independent experiments.
10
B10 CELLS INHIBIT EAE
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FIGURE 7. B10 cells alter T cell cytokine profiles, but not T cell proliferation. Purified splenic CD1dhighCD5+ or CD1dlowCD52 B cells from mice with
EAE (day 28) were stimulated with agonistic CD40 mAb for 48 h, with LPS added during the final 5 h of culture. A, B10 cell effects on T cell proliferation.
CFSE-labeled TCRMOG CD4+ T cells were cultured alone or with CD40/LPS-stimulated CD1dhighCD5+ or CD1dlowCD52 B cells in the presence of
MOG35–55 (25 mg/ml) for 72 h. After 72 h, the cultured cells were stained for CD4 and Thy1.1 expression and analyzed for CFSE dilution by flow
cytometry. Representative frequencies of dividing CFSE-labeled cells are shown (gated on CD4+Thy1.1+CFSE+ cells). Bar graphs (left) indicate CFSE
geometric mean fluorescence of the entire histogram, which is inversely proportional to cell divisions. Bar graphs (right) indicate mean (6SEM)
frequencies of dividing TCRMOG CD4+ T cells (CD1dhighCD5+ B cell group, n; CD1dlowCD52 B cell group, N) from six mice in each group from two
pooled independent experiments. B, B10 cells alter CD4+ T cell cytokine production. TCRMOG CD4+ T cells were cultured with CD40/LPS-stimulated
CD1dhighCD5+ or CD1dlowCD52 B cells from wild-type or IL-102/2 mice with EAE (day 28) in the presence of MOG35–55 (25 mg/ml) for 72 h, with PMA,
ionomycin, and BFA added during the final 5 h of culture. For negative controls, TCRMOG CD4+ T cells were cultured with CD40/LPS-stimulated CD1dlow
CD52 B cells in the presence of MOG35–55 (25 mg/ml) for 72 h, with BFA alone (without PMA/ionomycin) added during the final 5 h of culture. Cytokine
production by TCRMOG CD4+ T cells was determined by intracellular cytokine staining with flow cytometry analysis. Numbers indicate percentages of
T cells within the indicated gates among total CD4+ T cells. Bar graphs indicate mean (6SEM) percentages of cytokine-producing CD4+ T cells from six
mice in each group from two pooled independent experiments. C, Representative IL-10R expression (thick line) by splenic CD11c+ DCs, CD11bhigh
The Journal of Immunology
CD5+ spleen B cells containing 1.2 3 105 B10 cells from naive
mice reduced EAE severity, whereas 1.0 3 106 CD1dhighCD5+
spleen B cells containing only 1.3 3 105 B10 cells from mice
with EAE significantly reduced EAE severity (Fig. 5C). The transfer of 1.0 3 106 CD1dhighCD5+ spleen B cells from mice with
EAE containing 3.9 3 105 in vitro matured B10 + B10pro cells
was able to dramatically reduce EAE severity. Thereby, the identification of Ag-specific B10 cells may significantly reduce the
number of adoptively transferred B10 cells needed for inhibiting
EAE in vivo. Importantly, the adoptive transfer of IL-102/2
CD1dhighCD5+ B cells did not affect EAE responses under any
conditions tested. Thus, in addition to their preprogrammed ability
to rapidly proliferate in response to external stimuli (21) and
produce IL-10 (17), the size of the endogenous B10 and B10pro
cell pool is a critical factor for regulating the magnitude of acute
inflammation and the induction of autoimmunity.
B10 cells regulate T cell-mediated inflammatory responses and
EAE through IL-10–dependent mechanisms (12, 17). The current
study demonstrated that B10 cells did not directly regulate T cell
proliferation (Fig. 7A), but significantly reduced CD4+ T cell
IFN-g and TNF-a production during in vitro assays (Fig. 7B).
By contrast, non-CD1dhighCD5+ B cells were not able to influence
CD4+ T cell IFN-g and TNF-a production. Lampropoulou et al. (44)
have shown that tissue culture supernatant fluid from LPS-stimulated
spleen B cells does not suppress CD4+ T cell proliferation in
response to CD3 mAb stimulation in vitro, but is able to suppress
IFN-g secretion by T cells stimulated with CpG-stimulated DC.
Tregs isolated from the CNS are also able to suppress T cell IFN-g
production in response to MOG35–55 (45). Furthermore, B10 cells
may also downregulate the ability of DCs to act as APCs and
thereby indirectly modulate T cell proliferation (Fig. 7). Consistent with these findings, others have found that IL-10 suppresses
the proliferation of Ag-specific CD4+ T cells by inhibiting the
Ag-presenting capacity of monocytes and DCs (46) as well as
inhibiting proinflammatory cytokine production by monocytes
and macrophages (47). Lampropoulou et al. (44) have also shown
that tissue culture supernatant fluid from LPS-stimulated spleen
B cells is able to suppress T cell activation by CpG-stimulated
DCs through IL-10–dependent pathways. IL-10 was initially associated with Th2 cells and was described to inhibit Th1 cytokine
production (48–50). However, IL-10 is not only involved in the
inhibition of Th1 polarization, but also prevents Th2 responses
and exerts anti-inflammatory and suppressive effects on most hematopoietic cells. IL-10 produced by monocytes and cells other
than T cells is required to maintain Treg-suppressive function and
to maintain expression of the FoxP3 transcription factor in mice
with colitis (51). However, the adoptive transfer of in vitro matured spleen B10 + B10pro cells from mice with EAE significantly
reduced the number of Tregs within the CNS, in addition to reducing the number of inflammatory foci (Fig. 6). It is thereby
unlikely that IL-10 produced by B10 cells contributes significantly
to Treg maintenance during inflammation in vivo. However, future
macrophages, CD19+ B cells, CD4+ T cells, and CD8+ T cells from wild-type mice before or 7 d after MOG35–55 immunization. Gray histograms represent
isotype-matched control mAb staining. Bar graphs indicate average mean linear fluorescence intensities (6SEM) of IL-10R expression by each cell type
from six mice in each group from two pooled independent experiments. D, B10 cells inhibit the ability of DCs to activate CD4+ T cells. Splenic CD1dhigh
CD5+ or CD1dlowCD52 B cells from mice with EAE (day 28) were purified by cell sorting, and cultured with agonistic CD40 mAb for 48 h, with LPS
added during the final 5 h of culture. Purified splenic DCs from mice with EAE (day 10) were cultured alone or with CD40/LPS-stimulated CD1dhighCD5+
or CD1dlowCD52 B cells in the presence of MOG35–55 (25 mg/ml) for 72 h. DCs purified from the B cell cocultures were then cultured with TCRMOG CD4+
T cells for 72 h, or the T cells were cultured alone, with CD4 and Thy1.1 expression and CFSE dilution analyzed by flow cytometry. Mean frequencies of
dividing CFSE-labeled CD4+Thy1.1+CFSE+ cells are indicated (6SEM) in each histogram from six mice in each group from two pooled independent
experiments. Bar graphs indicate CFSE geometric mean fluorescence of the entire histogram, which is inversely proportional to cell divisions. Significant
differences between groups indicated; pp , 0.05
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roles in controlling EAE initiation and late-phase immunopathogenesis.
The timing of differential B10 cell and Treg expansion within
tissues parallels their importance during disease initiation and
late-phase EAE pathogenesis. B10 cell and CD1dhighCD5+ B cell
numbers expanded significantly (70% increase) by 7 d after
MOG35–55 immunization, decreased, and expanded to even higher
levels during the course of disease progression, with maximum
numbers (130% increase) accumulating as disease resolved (Fig.
1A, 1B). IL-10 production by spleen B cells and blood B10 cell
numbers followed a similar course of expansion, contraction, and
expansion (Figs. 1C, 4C), whereas lymph node B10 cell numbers
expanded most significantly during disease resolution (Fig. 4B).
By contrast, Treg numbers did not increase following MOG35–55
immunization, but increased gradually with EAE progression (Fig.
2C). On a relative frequency basis, B10 cells were 4 times more
prevalent in the CNS than Tregs before MOG35–55 immunization,
and their numbers remained constant except during disease resolution (Fig. 4A). By contrast, Tregs were 19-fold more prevalent in
the CNS on day 28 than B10 cells, whereas spleen B10 cell and
Treg frequencies were equal throughout the course of EAE (Fig.
4D). The absence of Treg expansion during EAE initiation provides the likely mechanistic explanation for why B cell depletion
by CD20 mAb or B10 cell depletion by CD22 mAb before
MOG35–55 immunization exacerbated EAE (Fig. 3B) (12). Reciprocally, the dramatic expansion of Tregs within the CNS during
EAE progression explains why the removal of B cells or B10 cells
at this time does not enhance disease pathogenesis. This was
confirmed when denileukin diftitox-induced Treg depletion had
no effect on disease initiation, but only exacerbated late-phase
disease (Fig. 3D). Denileukin diftitox treatment also suppresses
active EAE in rats when given early, but results in lethal disease if
given later (41). In contrast to the current study in wild-type mice,
it has been previously reported that IL-10–producing B cells
contribute to EAE recovery in mice that are genetically B cell
deficient (11). However, this is explained by the finding that
B cell deficiency delays the emergence of Tregs and IL-10 in
the CNS during EAE (42). Thereby, the relative balance between
B10 cell and Treg numbers in wild-type mice during the course of
EAE has dramatic effects on disease outcome.
Ag-specific B10 cell expansion is required to elicit B10 cell regulatory functions, and a diverse repertoire of B cell Ag receptors is
required for B10 cell development (12, 17, 21). Thereby, the ability
of B10 cells to rapidity expand during EAE initiation and to
quickly inhibit disease severity suggests that a sufficient pool of
Ag-specific B10 cells exists naturally that are rapidly mobilized to
inhibit inflammation. This is supported by adoptive transfer
experiments in which MOG35–55-primed CD1dhighCD5+ B cells,
but not naive CD1dhighCD5+ B cells, inhibited EAE development
(Fig. 5). The spleen of adult C57BL/6 mice normally contains
0.5–1.0 3 106 B10 cells and 3.5–4.5 3 106 B10 + B10pro cells
(43). Remarkably, the adoptive transfer of 1.0 3 106 CD1dhigh
11
12
Acknowledgments
We thank Dr. Karen Haas for helpful suggestions and Dr. Vijay Kuchroo for
providing mice for this study.
Disclosures
T.F.T. is a paid consultant for MedImmune and Angelica Therapeutics. The
authors have no other financial conflicts of interest.
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CD22 mAb treatment preferentially depleted spleen B10 cells
and exacerbated EAE disease severity (Fig. 3A, 3B). Because the
CD22 mAb used in this study blocks CD22 ligand binding, it is
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13